In order to explore the nature of a molecule that could be a receptor for a specific ligand (such as physiologically active substance, drug or antibody that acts upon binding to a cell composing, macromolecular component, e.g., protein or nucleic acid), usually, several techniques selected from various existing technologies are used in combination. For example, various techniques such as a technique of identifying an unknown receptor by such as photoaffinity labeling using a drug derivative or the like as a ligand; a technique of the intracellular localization of the receptor; a technique of isolating/purifying the receptor and examining its nature; or a technique of elucidating the binding site within the receptor (complex) molecule with a higher spatial resolution must be used jointly as the purpose demands. Even when one ligand is to be investigated, it is necessary to prepare separately a number of ligand derivatives suitable for individual techniques, which imposes a tremendous burden.
As a method of identifying those components to which a specific drug binds, affinity labeling is known. In this method, a ligand derivative to which a fluorescent dye or radioactive isotope has been added is photo-crosslinked to a target. Subsequently, information such as the molecular weight or amino acid sequence of the resultant labeled molecule is obtained using electrophoresis or various chromatographies.
For isolation and purification of a substance which will be a target binding partner for a ligand (such as receptor), a series of techniques called affinity purification is often used. When the target is a protein or a complex thereof, the classical chromatography is usually used in which resin beads immobilizing a ligand by covalent bond are packed in a column; a raw solution containing the target material is applied to the column; and the bound fractions alone are dissociated and eluted. However, when the target is a large-sized membrane fraction or non-adherent cell each of which is difficult to apply to the column, a batch method is also used in which similar resin beads or magnetic beads are utilized to collect the target substance by centrifugation or magnetism. In particular, when a target substance is to be separated after binding to a protein that is embedded in the membrane (such as intracellular organelle or non-adherent cell), a ligand directly immobilized on the surfaces of resin or magnetic beads is difficult to contact the target substance in many cases. In order to solve this problem, a spacer consisting of long straight carbon chain is inserted between the beads and the ligand. However, when a long carbon chain is used, hydrophobicity often increases. This makes it highly possibility that the binding of the target substance by the ligand does not necessarily reflect their specificity. For alleviation of this problem, it is desirable to use a spacer capable of always retaining a long distance.
On the other hand, in order to microscopically indicate the intracellular or extracellular localization site of a ligand-binding protein, a part or the whole of the protein prepared by a biochemical or molecular biological technique is used to prepare a specific antibody. Then, the localization (site) of the protein is elucidated by immunofluorescence or immunoelectron microscopy using the antibody. Further, for searching the binding site within the target molecule (complex), conventionally, structural data are collected by those means capable of obtaining atomic resolution, e.g., purifying and crystallizing the receptor/ligand complex and subjecting the crystal to X ray diffraction.
Although comprehensive methods to explore a large number of materials have been invented (e.g., use of robots), basically, researchers have no choice but to combine these techniques and proceed step by step in order to achieve their initial purpose. Regardless of what processes are employed, it is necessary to prepare individual ligands suitable for selected techniques. This always imposes a considerable burden in terms of labor, time and cost.
[Patent Document 1] Japanese Unexamined Patent Publication No. 2005-291836